Studies On Optimization Of Fermentation Conditions And Purification Of Anti-fungal Lipopeptide Produced By Bacillus Subtilis E1R-j

Bacillus subtilis strains can produce a broad spectrum of antibiotic compounds includinglipopeptides, peptides, phospholipids, alkenes, amino acids, and nucleic acids and so on. Theyare used to inhibit the growth of fungi, bacteria, virus and thallus, thus having important valueon the bio-control of plant dieases. However, it is the main bioactive substance-lipopeptide,that play an important role in controlling many plant dieases. But it is hard to applicatebecause of its low production. B.subtilis E1R-j strain, previously isolated from the root ofwheat, was used for producing lipopeptides. Former experiments have showed that itscell-free supernatant has good inhibitory effect not only on many kinds of fungi underlaboratory test such as Gaeumannomyces graminis var.tritici, Valsa mali var. mali, Botrytiscinerea and Alternarin solani and so on, but also on Gaeumannomyces graminis var.tritici infield experiment. In this research, the fermentation condition of lipopeptide produced by B.subtilis E1R-j strain was optimized by using single factor test conbioned with responsesurface methodology(RSM). Then, the substance was purified with reverse-phasechromatography, besides, the mass of bioactive substance was identified by means of massspectrum(MODI-TOF-TOF/MS) and finally its characterization was studied. The results asfollows:1. In Landy medium, the factors and its levels influencing the production of lipopeptideproduced by E1R-j strain, was determined by using single-factor test, and the optimization offermentation conditions was approached by using RSM. The optimum fermentationconditions were as follows: temperature was40℃,pH in Landy medium was9.0,inoculationwas3.0%,medium volume was50mL,rotary speed was200r/min.Under the new condition,the concentration of crude lipopeptide was1.39g/L, which was more than twice as much ofbefore-0.55g/L.2. There were four kinds of purified components:P₁, P₂, P₃and P₄.Their antifungalactivity and antifungal spectrum were different. P₁had good inhibitory effect onGaeumannomyces graminis var.tritici and Alternarin solani;P₂has good inhibitory effect on Gaeumannomyces graminis var.tritici;P₃has the best inhibitory effect on Gaeumannomycesgraminis var.tritici, Valsa mali var. mali, Botrytis cinerea and Alternarin solani; P4had goodinhibitory effect on Gaeumannomyces graminis var.tritici and Alternarin solani.3. The result of Mass spectrum identification showed that molecular mass of P2was915.5576Da,molecular mass of P₃were1477.846Da,1491.898Da,1505.960Da and1513.964Da,1527.992Da, and molecular mass of P₄were901.6007Da,915.6376Da and902.5656Da,916.5887Da。4. The research showed that the inhibitory effect of P₃had insignificant difference afterbeing treated by different temperature between25℃and80¹21℃,that is to say,it was notsensitive to temperature.Besides,P3was also not sensitive to Protease K and it can remainbioactivity in different organic solvent(methanol,acetonitrile and acetone).But its bioactivitywas highest in methanol.